The ob gene, an adipose specific gene believed to encode a satiety factor that regulated food intake and energy expenditure, has recently been discovered in mice. Two separate mutations in the mouse ob gene result in the development of morbid obesity. We have sequenced the coding region of the human ob gene and do not detect any mutations in the gene in either lean or obese subjects. However, we did find that the amount of ob gene message in human adipocytes is strongly correlated with the body mass index of the tissue donor. It is therefore plausible that the ob gene in humans codes for a satiety signal that informs the brain of the size of adipose tissue depot. The goal of this research proposal is to elucidate the mechanism(s) that regulate the ob gene satiety signal pathway in humans. In preliminary experiments we have been able to detect the ob protein in human adipocytes. Therefore, we will demonstrate that the rate of synthesis of ob protein is correlated with the amount of message, and that there is more ob protein in obese adipocytes compared to lean. We will further demonstrate that the ob protein is secreted into the blood and that the circulating concentration of the satiety signal is higher in obese individuals. The mechanisms that regulate ob signal synthesis and secretion from the adipocyte will also be evaluated. Our preliminary data suggest that the lipid content of the adipocyte is one factor that regulates the amount of ob gene mRNA. To further test this hypothesis, the lipid content of the adipose tissue will be altered in vivo through weight loss, weight gain, and fasting. The amount of ob mRNA, as well as tissue and circulating ob protein, will be quantitated to determine the response of the ob signal system to global changes in the adipose tissue. At the cellular level, isolated adipocytes in culture will be exposed to fatty acids (both long and short chain), hormones (insulin, norepinephrine, ob protein) and pharmacological agents (isoproterenol, troglitazone) to evaluate their ability to regulate ob signal output. The effect of these agents on both transcription and translation will be assessed. In addition, we will determine if change in amount of ob mRNA are due to transcriptional or post-transcriptional mechanisms, or both. Should transcriptional regulation of ob signal output be an important mechanism, we will determine what transcriptional elements are involved. The completion of the studies described in this proposal will further our understanding of the ob signal system and the mechanisms that regulate energy storage in humans. This effort is possible because we have access to a steady supply of adipose tissue from healthy lean, as well as obese, individuals through our unique outpatient adipose tissue biopsy program. We believe that the knowledge obtained through the proposed studies will ultimately lead to improvement in the management or prevention of obesity.